Puffer fish TRPV1 inhibitory peptide as well as preparation method and application thereof

Abstract

The invention relates to a puffer fish TRPV1 inhibitory peptide as well as a preparation method and application thereof. The amino acid sequence of the peptide is LDIF. The preparation method comprises the following steps: 1, performing enzymolysis on the fish skin of the puffer fish, performing enzyme deactivation, then screening out polypeptide with the molecular weight not greater than 1kDa from enzymatic hydrolysate, and performing freeze-drying to obtain the puffer fish skin enzymolysis polypeptide; 2, performing mass spectrum determination on the puffer fish skin enzymolysis polypeptide, analyzing a mass spectrum determination result by adopting mass spectrum analysis software, and screening out a plurality of non-repeated polypeptide sequences under the screening conditions that the credibility-10lgP is greater than 20 and the number of amino acids is less than 10; 3, carrying out molecular docking on the screened polypeptide sequence and the TRPV1 receptor through software, and screening the polypeptide sequence with strong binding capacity with the TRPV1 receptor; and 4, performing solid-phase synthesis on the screened polypeptide sequence to obtain the polypeptide LDIF. According to the invention, the puffer fish skin is used as a raw material to obtain the polypeptide with a good inhibition effect on TRPV1, and the polypeptide can be used for preparing a skin care product for relieving sensitive skin.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and particularly relates to a pufferfish TRPV1 inhibitory peptide, a preparation method thereof, and an application thereof. Background Art

[0002] Sensitive skin specifically refers to a highly reactive state that occurs under physiological or pathological conditions of the skin, mainly occurring on the face. The clinical manifestations are that when the skin is stimulated by physical, chemical, mental and other factors, subjective symptoms such as burning, stinging, itching and tightness are likely to appear, with or without objective signs such as erythema, scaling, telangiectasia, etc. It is pointed out in the "Chinese Expert Consensus on the Diagnosis and Treatment of Sensitive Skin" that the incidence rate of women is generally higher than that of men, about 36.1%. Under the interaction of external and internal factors such as environmental climate change, improper use of cosmetics, stress and emotions, eating habits, etc., the occurrence of sensitive skin will be caused. The formation of sensitive skin is a complex and delicate process, involving multiple factors such as the destruction of the skin barrier, the abnormal excitation of neurovascular, and the activation of immune inflammation.

[0003] When the skin barrier function is damaged, it will activate the transient vanilloid receptor (TRPV1). TRPV1 is a multimodally activated, calcium-permeable non-selective cation channel that participates in the conduction of sensitive-related sensory symptoms. When the TRPV1 receptor is activated, the TRPV1 channel causes action potentials, resulting in a large influx of Ca 2+ ions, causing symptoms such as burning, stinging and itching in sensitive skin. The activation of TRPV1 can also promote the release of substance P, vasoactive intestinal peptide, and neurotensin in the local skin. It will also cause keratinocytes (HaCaT) and mast cells near the sensory nerve endings to release interleukin-23 (IL-23) and interleukin-31 (IL-31), and antigen-presenting cells and T cells are subsequently activated, triggering skin immune and inflammatory responses. Endothelin (ET-1) is a polypeptide substance that causes vasoconstriction and vasodilation, mainly synthesized by various cells such as endothelial cells, neuronal cells, macrophages, etc. When vascular endothelial cells are stimulated, the secretion of ET-1 increases significantly. ET-1 can induce the up-regulation of intercellular cell adhesion molecule (ICAM-1) in endothelial cells, promote the release of inflammatory mediators, and lead to vascular dysfunction. Therefore, it is of great significance to find safe and effective TRPV1 activity inhibitors.

[0004] At present, artificially synthesized TRPV1 antagonists include trans-4-tert-butylcyclohexanol, asycropt, capsaicin, etc.; in addition, many extracts with TRPV1 antagonistic effects obtained from plants have been confirmed to have the effect of accelerating the recovery of skin barrier damage.

[0005] Natural product active peptides have a variety of functions in human metabolism and physiological regulation, with good biocompatibility and extremely high safety. They are one of the most popular research topics and functional factors with great development prospects in the current international food and cosmetics industries. Peptide TRPV1 antagonists are a research hotspot in the development of soothing active substances in recent years. The APHC peptide from sea anemone is the first reported peptide antagonist that binds to the TRPV1 channel and shows strong inhibitory effects on capsaicin (CAP)-induced TRPV1 activation. In vivo studies have shown that the APHC peptide has significant analgesic effects in different pain models. Kang et al. developed a new TRPV1-targeted peptide (TIP) and found that TIP can effectively inhibit CAP-induced calcium influx and TRPV1 activation. TIP reduces UV-induced erythema and the expression of inflammatory factors in human skin in vivo, providing a treatment method for UV-induced inflammation and photoaging. However, there is very little research on TRPV1 inhibitors from fish at present. Summary of the Invention

[0006] The purpose of the present invention is to provide a pufferfish TRPV1 inhibitory peptide, its preparation method and application. Using pufferfish skin as raw material, the amino acid sequence of the pufferfish TRPV1 inhibitory peptide is obtained, and the prepared pufferfish TRPV1 inhibitory peptide has a good inhibitory effect on TRPV1 and can be used to prepare skin care products for soothing sensitive skin.

[0007] To achieve the above purpose, the present invention discloses a pufferfish TRPV1 inhibitory peptide, and its amino acid sequence is: LDIF.

[0008] The present invention also discloses a preparation method of the above pufferfish TRPV1 inhibitory peptide, which includes the following steps: S1. Extraction of pufferfish skin enzymatically hydrolyzed polypeptide Use alkaline protease to enzymatically hydrolyze pufferfish skin. After the enzymatic hydrolysis is completed, inactivate the enzyme, and then screen out polypeptides with a molecular weight not greater than 1 kDa from the enzymatic hydrolysate, and freeze-dry to obtain pufferfish skin enzymatically hydrolyzed polypeptide; S2. Sequence identification and preliminary screening Perform mass spectrometry on the pufferfish skin enzymatically hydrolyzed polypeptide. The results of the mass spectrometry are analyzed using mass spectrometry analysis software, and multiple non-repeating polypeptide sequences are screened out with the screening conditions of confidence -10lgP>20 and the number of amino acids <10; S3. Virtual screening Use software to perform molecular docking of the polypeptide sequences screened in step S2 with the TRPV1 receptor, and screen out polypeptide sequences with strong binding ability to the TRPV1 receptor; S4. Polypeptide synthesis Solid-phase synthesize the polypeptide sequences screened in step S3 to obtain the pufferfish TRPV1 inhibitory peptide.

[0009] Preferably, it further includes step S0 carried out before step S1. Step S0 is the pretreatment of fish skin, specifically: first cut the fish skin of pufferfish into small pieces, and then perform freeze-drying treatment on the cut small pieces of fish skin; then soak the freeze-dried fish skin in NaCl solution and continuously stir for 12 - 36h, changing the NaCl solution every 6 - 12h. The mass concentration of the NaCl solution is 6 - 12%; after the fish skin soaking is completed, wash it with distilled water and filter it with nylon yarn, repeating 3 - 5 times.

[0010] Preferably, in step S1, the conditions for alkaline protease to enzymatically hydrolyze the fish skin of pufferfish are: solid-liquid ratio 1:10, enzyme addition amount 8000 U / g, enzymatic hydrolysis temperature 50°C, enzymatic hydrolysis pH value 9.0, enzymatic hydrolysis time 4h, enzymatic hydrolysis temperature 100°C.

[0011] Preferably, in step S1, the enzymatic hydrolysate is first microfiltered through a ceramic membrane, and then ultrafiltered through an ultrafiltration membrane with a molecular weight cut-off of 1 kDa to separate polypeptides with a molecular weight not greater than 1 kDa.

[0012] Preferably, the specific operation of mass spectrometry determination of the enzymatically hydrolyzed polypeptide of pufferfish skin in step S2 is as follows: dissolve the enzymatically hydrolyzed polypeptide of pufferfish skin in solvent A to obtain sample peptides; load 1 μL of sample peptides onto a 25 cm analytical column, and start separation from 2% buffer B with a 60 min gradient, gradually increasing to 35% in 47 min, increasing to 100% in 1 min, and maintaining for 12 min; the column flow is maintained at 300 nL / min, and the column temperature is 40°C; the electrospray voltage is set to 2 kV; the mass spectrometer operates in data-dependent acquisition mode and automatically switches between MS and MS / MS modes; a full-scan mass spectrum is obtained on an Orbitrap with a resolution of 70,000; the automatic gain control target is 3e6, and the maximum injection time is 50 ms; select precursor ions to enter the collision cell for high-energy collision dissociation; fragmentation, the normalized collection energy is 28%; the MS / MS resolution is set to 17,500, the automatic gain control target is 1e5, the maximum injection time is 45 MS, and the dynamic exclusion time is 30 s; wherein, solvent A is an aqueous solution of formic acid with a mass concentration of 0.1%, and buffer B is an aqueous solution containing ACN with a mass concentration of 80% and FA with a mass concentration of 0.1%.

[0013] Preferably, the crystal structure of the TRPV1 receptor is 8GFA.

[0014] In addition, the present invention also discloses the application of the above-mentioned pufferfish TRPV1 inhibitory peptide in the preparation of skin care products.

[0015] Preferably, the mass concentration of the pufferfish TRPV1 inhibitory peptide in the skin care product is not less than 400 μM.

[0016] The present invention has the following beneficial effects: The present invention uses puffer fish skin as a raw material to obtain the amino acid sequence of puffer fish TRPV1 inhibitory peptide, and the prepared puffer fish TRPV1 inhibitory peptide has a good inhibitory effect on TRPV1. It can be used to prepare skin care products for soothing sensitive skin, providing a good candidate compound for the development of skin care products for sensitive skin. Description of the Drawings

[0017] Figure 1 Effect of different concentrations of T14 on the activity of RAW 264.7 cells.

[0018] Figure 2 Inhibitory effect of different concentrations of T14 on the NO content.

[0019] Figure 3 Fluorescence intensity of Fluo-4 AM-loaded HaCaT cells responding to CAP at different concentrations of T14.

[0020] Figure 4 Effect of T14 action time on the increase in intracellular Ca 2+ concentration induced by CAP.

[0021] Figure 5 3D view of the best docking conformation of T14 with TRPV1.

[0022] Figure 6 For Figure 5 Enlarged schematic view of part A in

[0023] Figure 7 2D map of the interaction between T14 and TRPV1.

[0024] Figure 8 Inhibitory effect of different concentrations of T14 on the expression level of ET-1.

[0025] Figure 9 Inhibitory effect of different concentrations of T14 on the expression level of ICAM-1.

[0026] Figure 10 Cytokine array map of RAW 264.7 cells.

[0027] Figure 11 Heat map of the gene expression of 40 cytokines.

[0028] Note: Figure 10 Each pair of points in represents 2 parallels of each cytokine or chemokine. Detailed Description of the Invention

[0029] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and embodiments.

[0030] The present invention discloses a pufferfish TRPV1 inhibitory peptide, whose amino acid sequence is: LDIF, as shown in SEQ ID NO: 1 in the sequence listing.

[0031] The preparation method of the above-mentioned pufferfish TRPV1 inhibitory peptide comprises the following steps: S0. Pretreatment of fish skin The pufferfish used as the test raw material in this case is Takifugu obscurus from Zhangpu, Fujian. First, cut the fish skin of the pufferfish into small pieces with a meat cutter, and then freeze-dry the cut fish skin to remove the moisture in the fish skin, and store it in vacuum packaging. Then soak the freeze-dried fish skin in a NaCl solution with a mass concentration of 9%, and continuously stir for 24 h, replacing the NaCl solution every 12 h. After the fish skin soaking is completed, wash it with distilled water and filter it with nylon yarn, and repeat 3 times to remove non-collagen proteins.

[0032] S1. Extraction of pufferfish skin enzymatically hydrolyzed polypeptide Use alkaline protease (B8360, Solarbio) to enzymatically hydrolyze the fish skin of the pufferfish. The optimal enzymatic hydrolysis conditions are: solid-liquid ratio 1:10, enzyme dosage 8000 U / g, enzymatic hydrolysis temperature 50 °C, enzymatic hydrolysis pH value 9.0, enzymatic hydrolysis time 4 h, and enzyme deactivation temperature 100 °C.

[0033] After the enzymatic hydrolysis is completed, inactivate the enzyme for 10 min. First, microfilter the enzymatic hydrolysate through a ceramic membrane, and then separate it through an ultrafiltration membrane with a molecular weight cut-off of 1 kDa, and screen out polypeptides with a molecular weight not greater than 1 kDa. After vacuum freeze-drying, store it at -20 °C to obtain the pufferfish skin enzymatically hydrolyzed polypeptide.

[0034] S2. Sequence identification and preliminary screening Perform mass spectrometry on the pufferfish skin enzymatically hydrolyzed polypeptide. The results of the mass spectrometry are analyzed using mass spectrometry analysis software, and multiple non-repeating polypeptide sequences are screened out with the screening conditions of confidence -10lgP > 20 and the number of amino acids < 10.

[0035] Specifically, the polypeptide sequence was identified by Nano-HPLC-MS / MS. The polypeptide was redissolved in solvent A (A: 0.1% formic acid aqueous solution) to obtain the sample peptide, and analyzed by an Orbitrap Q-Exactive Plus and EASY-nanoLC 1200 system (Thermo Fisher Scientific, MA, USA). 1 μL of the sample peptide was loaded onto a 25 cm analytical column (inner diameter 75 μm, resin 1.9 μm (Dr Maisch)), and separated with a 60 min gradient starting from 2% buffer B (80% ACN (acetonitrile), 0.1% FA (formic acid)), gradually increasing to 35% in 47 min, increasing to 100% in 1 min, and maintaining for 12 min. The column flow was maintained at 300 nL / min, and the column temperature was 40 °C. The electrospray voltage was set to 2 kV. The mass spectrometer was operated in data-dependent acquisition (DDA) mode and automatically switched between MS and MS / MS modes. A full-scan mass spectrum (m / z 200 - 1800) was obtained on the Orbitrap at 70,000 resolution. The automatic gain control (AGC) target was 3e6, and the maximum injection time was 50 ms. The precursor ions were selected to enter the collision cell for high-energy collision dissociation (HCD) fragmentation, and the normalized collision energy was 28%. The MS / MS resolution was set to 17,500, the automatic gain control (AGC) target was 1e5, the maximum injection time was 45 ms, and the dynamic exclusion time was 30 s.

[0036] The polypeptide sequence was identified by nano-HPLC-MS / MS. In the peptide results, the -10 lgP index represents the confidence level of the corresponding spectrum identification. The larger the value, the better the matching result. According to the mass spectrometry analysis results, peptides with a -10lgP > 20 for spectrum identification and the number of amino acids < 10 were screened. A total of 190 polypeptides were preliminarily screened.

[0037] S3. Virtual screening The polypeptide sequences screened in step S2 were molecularly docked with the TRPV1 receptor through software to screen out the polypeptide sequences with strong binding ability to the TRPV1 receptor.

[0038] Specifically, based on the analysis results of mass spectrometry, peptides were initially screened according to the confidence level of spectral identification -10 lgP and the number of amino acids. TRPV1 (PDB: 8GFA) was used as the receptor protein, and its 3D structure was downloaded from the RCSB PDB database (https: / / www.rcsb.org). Before virtual screening, the MOE 2022 software was used to remove unnecessary water molecules from the receptor protein, add hydrogen, add charges, and optimize the energy; the polypeptide was used to draw small molecules as ligands using the Discovery Studio 2019 Client software to determine the active pocket of TRPV1, and the position of the active pocket (X: 108.603; Y: 79.704; Z: 88.011). Molecular virtual screening was carried out using DOCK 6.9, and the lead peptide LDIF (T14) was screened out according to the Grid Score value and binding mode.

[0039] S4. Peptide synthesis The polypeptide sequence (LDIF (T14)) screened in step S3 was solid-phase synthesized to obtain the pufferfish TRPV1 inhibitory peptide.

[0040] Next, the lead peptide LDIF screened was experimentally verified in terms of cell activity, inhibitory effect on NO content, inhibitory effect on CAP-induced Ca 2+ influx in HaCaT cells, and inhibitory effect on the irritant response of HUVEC cells.

[0041] I. Test cell culture RAW 264.7 and HUVEC cells were purchased from the Peking Union Medical College Cell Bank, and HaCaT cells were purchased from the Cell Bank of the Kunming Institute of Zoology, Chinese Academy of Sciences. RAW 264.7 and HaCaT cells were cultured in DMEM (Gibco, C11995500BT) complete medium containing 10% FBS (Sigma, F8687) and 1% penicillin-streptomycin (Beyotime, C0222). HUVEC cells were cultured in DMEM (Gibco, C11995500BT) complete medium containing 1% glutamine, 1% non-essential amino acids (NEAA), 2% sodium pyruvate, 10% FBS (Sigma, F8687), and 1% penicillin-streptomycin (Beyotime, C0222).

[0042] II. Determination of cell activity The MTT (MTS, Promega; PMS, Sigma) method was used to determine the effect of the polypeptide on the proliferation of Raw 264.7 cells. Specifically, the activity of T14 on RAW 264.7 cells in the concentration range of 100 - 800 μM was determined by the MTT method, and a blank control group (CK) was set. The results are as Figure 1As shown. From Figure 1 It can be seen that the cell viability of T14 is the lowest at 200 μM, which is 86%. When the cell viability is above 80%, it is considered non-toxic to cells. Therefore, T14 has no toxicity to RAW 264.7 cells at a concentration of 800 μM.

[0043] III. Determination of NO content RAW 264.7 cells were cultured in DMEM medium containing 10% FBS and 1% double antibody in a constant temperature incubator at 37°C and 5% CO2. RAW 264.7 cells in the logarithmic growth phase were taken, made into a suspension, and the cell density was adjusted to 2×10 5 cfu / mL, inoculated into a 24-well plate at 500 μL per well, and placed in the incubator for 8 - 12 h. The supernatant was aspirated, and cells were treated with T14 solutions at different concentrations (50, 100, 200, 400 μM) for 2 h, and then LPS (final concentration 1 μg / ml) (L8880, Solarbio) was added for co-incubation for 24 h; the NO expression level was detected according to the NO kit instructions, and the results are as Figure 2 shown (Note: Figure 2 In the group with the abscissa labeled "0" in, it represents the blank control, and the group labeled "LPS" represents the group with only LPS added. Figure 3 The abscissa labeling method of Figure 2 is similar, and will not be elaborated here).

[0044] From Figure 2 it can be seen that compared with the LPS group, the inhibition rate of T14 is 14.6% at 200 μM and 46.1% at 400 μM, and it can significantly inhibit the production of NO.

[0045] IV. Ca 2+ influx experiment HaCaT cells in the logarithmic growth phase were taken and inoculated into a 96-well black-wall transparent plate at 1×10 5 cfu / mL, cultured overnight, and the supernatant was discarded. 200 μM of T14 was added to each well in the 96-well plate and cultured for 2 h, 6 h, 12 h, and 24 h. Then, 100 μL of CAP was added to each well for stimulation for 30 min. A blank control group was set, and pictures were taken using an inverted fluorescence microscope. The taken pictures were processed using imaje.j, and the average fluorescence intensity was calculated.

[0046] An in vitro model was established by inducing HaCaT cells with CAP, and the Ca 2+ influx was used as an index to study the skin anti-allergy mechanism based on TRPV1. As Figure 3 - 4 shown, CAP can induce Ca 2+After the concentration increased and HaCaT cells were incubated with 200 μM of T14, it had a significant inhibitory effect on the intracellular Ca 2+ content induced by CAP, and it was time-dependent. When the incubation time was extended to 12 and 24 h, the inhibitory effect on the Ca 2+ concentration was more significant. After incubation for 24 h, the inhibition rate of T14 on the Ca 2+ fluorescence intensity was 73.1% ± 7.55%.

[0047] V. Interaction analysis between T14 and TRPV1 The docking result of T14 and TRPV1 is as Figure 5 - 7 shown. T14 binds in the cavity of TRPV1, forms a hydrogen bond with the residue THR550 (2.24 Å), and forms hydrophobic interactions with MET572 (4.26 Å), LEU553 (5.27 Å), TYR511 (4.59 Å), LEU515 (5.32 Å), LEU553 (5.27 Å), LEU577 (4.83 Å, 4.96 Å), ILE569 (4.78 Å), and forms sulfur bonds with MET568 (5.86 Å) and MET683 (5.95 Å); these three interaction forces contribute to the structural stability of the conjugate of the two.

[0048] VI. Stimulatory response of HUVEC cells HUVEC cells in the logarithmic growth phase were inoculated into 24-well plates at 2×10 5 cfu / mL, cultured overnight, and the supernatant was discarded. Different concentrations of T14 (50, 100, 200, 400 μM) were added to each well and cultured for 6 h in 24-well plates first, and then LPS (10 μg / mL) was added and co-cultured for 24 h. The cell culture supernatant was collected, and the expression levels of ET-1 and ICAM-1 were detected according to the instructions of the ELISA kit, and the inhibition rate was calculated. The calculation formula is as follows: Inhibition rate = (Expression level in LPS group - Expression level in polypeptide group) / (Expression level in LPS group - Expression level in negative control group) × 100% When vascular endothelial cells are stimulated by endogenous or exogenous stimuli, the secretion of ET-1 increases. The increase in ET-1 stimulates neutrophils and macrophages to release various pro-inflammatory factors, and the expression level of ICAM-1 will increase sharply, affecting vascular permeability, resulting in skin redness and swelling, causing vascular stimulatory reactions. As Figure 8As shown, when HUVEC cells were stimulated with 10 μg / mL of LPS for 24 h, the expression level of ET-1 increased from 2.05 pg / mL to 2.7 pg / mL. After pretreatment with T14 for 6 h and then co-cultured with LPS for 24 h, T14 dose-dependently decreased the expression level of ET-1. When T14 was 100 μM, the expression level of ET-1 decreased to 0.54 pg / mL, with an inhibition rate of 24.47%. When T14 was 400 μM, the expression level of ET-1 decreased to 2.2 pg / mL, and the inhibition rate reached 75%, showing a significant difference.

[0049] As Figure 9 shown, the expression level of ICAM-1 increased from 0.43 to 0.57 pg / mL under LPS stimulation. After pretreatment with T14, T14 dose-dependently decreased the expression level of ICAM-1. When T14 was 50 μM, the expression level of ICAM-1 decreased to 0.54 pg / mL, with an inhibition rate of 20.65%. When T14 was 400 μM, the expression level of ICAM-1 decreased to 0.50 pg / mL, and the inhibition rate reached 48.96%, showing a significant difference.

[0050] VII. Mouse Cytokine Array Analysis The protein expressions of 40 cytokines and chemokines in the culture supernatant of Raw 264.7 cells were measured using the protocol of the Proteome ProfilerTM Antibody Arrays Mouse Cytokine Array Panel A kit. The culture supernatants of each treatment group were collected, and 300 μL of the supernatant was used for the experiment for each array. At the beginning of the experiment, the reagents used were equilibrated to room temperature, and the membrane was blocked for 2 h. While blocking the membrane, the detection antibody mixture was added to the cell supernatant and incubated at room temperature for 1 h, and then incubated with the membrane at 4 °C overnight. After incubation, the membrane was washed 3 times with the washing solution for at least 10 min each time. After washing, HRP-conjugated streptavidin was added and incubated at room temperature for 2 h. After sufficient binding, it was washed again, and the membrane was exposed with chemiluminescent reagent. An array image showing chemiluminescent signals was obtained using a chemiluminescent imaging system, and the integrated optical density was analyzed using the image.j software through a densitometer.

[0051] From Figure 10 - 11It can be seen that, compared with the normal group, the LPS group led to a significant upregulation of the expression levels of cytokines such as G-CSF, GM-CSF, ICAM-1, IL-3, IL-6, TNF-α, CCL2, CCL3, CCL4, CXCL2, CCL5, and TIMP-1. After incubation with T14, the expression levels of cytokines such as G-CSF, GM-CSF, ICAM-1, IL-3, IL-6, IL-23, and TNF-α were significantly downregulated, and the expression level of TIMP-1 was upregulated; this indicates that T14 can inhibit the expression of pro-inflammatory factors produced by LPS-stimulated RAW 264.7 cells while increasing the expression level of TIMP-1.

[0052] Based on the above experiments, the present invention also discloses the application of the above-mentioned pufferfish TRPV1 inhibitory peptide (LDIF) in the preparation of skin care products, and the mass concentration of the pufferfish TRPV1 inhibitory peptide in the skin care products is not less than 400 μM. This skin care product is particularly suitable for sensitive skin.

[0053] As described above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A pufferfish TRPV1 inhibitory peptide, characterized in that, Its amino acid sequence is: LDIF.

2. The preparation method of the pufferfish TRPV1 inhibitory peptide according to claim 1, wherein, It includes the following steps: S1. Extraction of enzymatically hydrolyzed polypeptide from puffer fish skin Use alkaline protease to enzymatically hydrolyze the skin of puffer fish. After the enzymatic hydrolysis is completed, inactivate the enzyme, and then screen out polypeptides with a molecular weight not greater than 1 kDa from the enzymatic hydrolysate, and freeze-dry to obtain the enzymatically hydrolyzed polypeptide from puffer fish skin; S2. Sequence identification and preliminary screening Perform mass spectrometry determination on the enzymatically hydrolyzed polypeptide from puffer fish skin. The results of the mass spectrometry determination are analyzed using mass spectrometry analysis software, and the screening conditions are a confidence level of -10lgP>20 and the number of amino acids <10, and multiple non-repeating polypeptide sequences are screened out; S3. Virtual screening Use software to perform molecular docking of the polypeptide sequences screened in step S2 with the TRPV1 receptor, and screen out polypeptide sequences with strong binding ability to the TRPV1 receptor; S4. Polypeptide synthesis Solid-phase synthesize the polypeptide sequences screened in step S3 to obtain the puffer TRPV1 inhibitory peptide.

3. The preparation method according to claim 2, wherein: It also includes step S0 carried out before step S1. Step S0 is fish skin pretreatment, specifically: first cut the skin of puffer fish into small pieces, and then perform freeze-drying treatment on the cut small pieces of fish skin; then soak the freeze-dried fish skin in NaCl solution and continuously stir for 12-36 h, and change the NaCl solution every 6-12 h. The mass concentration of the NaCl solution is 6-12%; after the fish skin soaking is completed, wash it with distilled water and filter it with nylon gauze, and repeat 3-5 times.

4. The preparation method according to claim 2, characterized in that: In step S1, the conditions for alkaline protease to enzymatically hydrolyze the skin of puffer fish are: solid-liquid ratio 1:10, enzyme addition amount 8000 U / g, enzymatic hydrolysis temperature 50 °C, enzymatic hydrolysis pH value 9.0, enzymatic hydrolysis time 4 h, enzymatic hydrolysis temperature 100 °C.

5. The preparation method according to claim 2, characterized in that: In step S1, the enzymatic hydrolysate is first microfiltered through a ceramic membrane, and then ultrafiltered through an ultrafiltration membrane with a molecular weight cut-off of 1 kDa to separate polypeptides with a molecular weight not greater than 1 kDa.

6. The preparation method according to claim 2, wherein, The specific operation of mass spectrometry determination on the enzymatically hydrolyzed polypeptide from puffer fish skin in step S2 is as follows: dissolve the enzymatically hydrolyzed polypeptide from puffer fish skin in solvent A to obtain a sample peptide; load 1 μL of the sample peptide onto a 25 cm analytical column, and start separation from 2% buffer B with a 60 min gradient, gradually increase to 35% in 47 min, increase to 100% in 1 min, and hold for 12 min; the column flow rate is maintained at 300 nL / min, and the column temperature is 40 °C; the electrospray voltage is set to 2 kv; the mass spectrometer operates in the data-dependent acquisition mode and automatically switches between the MS and MS / MS modes; a full-scan mass spectrum is obtained on the Orbitrap with a resolution of 70,000; the automatic gain control target is 3e6, and the maximum injection time is 50 ms; select precursor ions to enter the collision cell for high-energy collision dissociation; fragment, and the normalized collection energy is 28%; the MS / MS resolution is set to 17,500, the automatic gain control target is 1e5, the maximum injection time is 45 MS, and the dynamic exclusion time is 30 s; among them, solvent A is an aqueous solution of formic acid with a mass concentration of 0.1%, and buffer B is an aqueous solution containing ACN with a mass concentration of 80% and FA with a mass concentration of 0.1%.

7. The preparation method according to claim 2, wherein: The crystal structure of the TRPV1 receptor is 8GFA.

8. Use of the pufferfish TRPV1 inhibitory peptide according to claim 1 in the preparation of skin care products.

9. The application according to claim 8, characterized in that: The mass concentration of the pufferfish TRPV1 inhibitory peptide in the skin care products is not less than 400 μM.

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